The highly dynamic structure of chromatin governs access of many proteins to the underlying DNA, affecting the regulation of numerous fundamental biological processes and developmental programs. The generation of a competent immune system requires the assembly of antibody and T cell receptor genes by a tightly regulated series of site-specific DNA recombination events that occur during lymphoid development. All these V(D)J rearrangement events are mediated by the same RAG1/RAG2 recombinase, yet the rearrangements themselves are lineage-specific and occur in a preferred temporal order, implying that the recombinase can only access a correctly opened locus. We recently showed that RAG2 contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated on lysine 4, and exhibits an even stronger preference for binding H3 that is concurrently trimethylated on lysine 4 and symmetrically dimethylated at arginine 2 (H3R2me2s/K4me3). Symmetrical dimethylation of histone H3 R2 has not been described in mammalian cells or model organisms. The goal of this proposal is to test the hypothesis that H3R2me2s alone or in combination with H3K4me3 is a novel epigenetic mark in mammalian cells, with a role in governing lymphoid development and perhaps many other cellular processes. First, we will develop and characterize antibodies to the individual histone H3R2me2s modification, and to the dual modification H3R2me2s/K4me3. We will also make a third antibody that recognizes H3R2me2s only in the absence of K4me3 (H3R2me2s/K4me3). We will use these antibodies to determine if these modifications are present at a bulk level in mammalian cells. Second, we will perform ChIP and quantitative PCR on chromatin from lymphoid cells to determine if there are locus, stage or lineage-specific distribution patterns of H3R2me2s and H3R2me2s/K4me3. We will also perform immunoprecipitation followed by Western blot analysis to determine if RAG2 PHD finger binds H3R2me2s/K4me3 in chromatin both in vitro and in vivo. Third, we will analyze the epigenomic landscape of H3R2me2s, H3R2me2s/K4me3, and H3R2me2s/K4me3 in mammalian cells in depth, using a combination of immunofluorescence and genome-wide chromatin immunoprecipitation on a range of cell types under various conditions. This analysis should enable us to determine functional correlates of these modifications. Fourth, we will analyze these modifications in model organisms (S. cerevisiae, S. pombe and Drosophila melanogaster), determining both the genome-wide localization pattern of the modifications and whether they are enriched in particular developmental stages, tissues, stages in the cell cycle, or in response to DNA damage. In summary, the proposed experiments should establish whether H3R2me2s is a novel epigenetic modification in mammalian cells and elucidate its possible functional roles. PUBLIC HEALTH RELEVANCE: DNA in human cells is packaged with proteins that can be modified to regulate how genes are expressed. We are studying a novel modification that we believe may play a role in generating a functional immune system and other aspects of cell development. The experiments proposed here should define the roles of this modification and lead to insights into its possible role in both normal development and human disease.